Dyes and Staining

 Dyes and Simple Staining

The dyes used to stain microorganisms have two features in common:

• ·     they have chromophore groups, (Chromophore is colored chemical substance that has ability to provide a color to dye)
• ·      they can bind with cells by ionic, covalent, or hydrophobic bonding.

Most dyes are used to directly stain the cell or object of interest. Some dyes (e.g., India ink and nigrosin) are used in negative staining, where the background but not the cell is stained; the unstained cells appear as bright objects against a dark background.

The most commonly used dyes bind cells by ionic interactions. These ionizable dyes may be divided into different classes based on the nature of their charged group.

 Stains can be broadly classified based on the nature of chromogen into:

1. Acidic stain (Anionic stain) with –ve charge  Nigrosin  Malchite green

2. Basic stain (Cationic stain) with +ve charge  Crystal violet  Methylene blue  Safrinin  Basic fuchsin

3.Neutral stain  Eosinate of methylene blue  Giemsa stain

1. Basic dyes- methylene blue, basic fuchsin, crystal violet, safranin, malachite green—have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids, proteins.

Because the surfaces of bacterial cells also are negatively charged, basic dyes are most often used in bacteriology.

2. Acidic dyes— Nigrosine, Picric acid, India ink, eosin, rose bengal, and acid fuchsin—possess negatively charged groups such as carboxyls (—COOH) and phenolic hydroxyls (—OH). Acidic dyes, because of their negative charge, bind to positively charged cell structures.

pH can alter the staining effectiveness of ionizable dyes because the nature and degree of the charge on cell components change with pH. Acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pH.

Dyes that bind through covalent bonds or because of their solubility characteristics are also useful. For instance, DNA can be stained by the Feulgen procedure in which the staining compound (Schiff’s reagent) is covalently attached to its deoxyribose sugars. Sudan III (Sudan Black) selectively stains lipids because it is lipid soluble but will not dissolve in aqueous portions of the cell.

 

Staining Techniques

1.   Simple staining

2. Differential Staining 

-Gram stain

-Acid-fast staining

3. Special staining

-Capsule staining

-Negative staining

-Endospore staining

-Flagella staining

4. Fungal staining

  

 Simple staining/Monochrome staining

Microorganisms can be stained by simple staining, in which a single dye (monochrome) is used to determine the size, shape, and arrangement of procaryotic cells. The bacterial smear is stained with a single reagent, which produces a sharp contrast between the organism and its background. 

Basic stains with a positively charged chromogen are preferred because bacterial nucleic acids and certain cell wall components has a negative charge and will strongly attract and bind to the cationic chromogen. Basic dyes like crystal violet, methylene blue, and carbolfuchsin are frequently used in simple staining

Simple staining is characterised by its simplicity and ease of use. The fixed smear is immersed in one stain for a short period of time, followed by washing off the excess stain with water, and blotting the slide dry. 

The purpose of simple staining is to elucidate the size, shape and arrangement of bacterial cells.

Bacilli and diplobacilli: Rod-shaped bacteria, purple
Cocci: spherical-shaped, bacteria, purple

 

Differential Staining 

Use two or more dyes  to differentiate between organisms according to their response to these dyes. Common differential staining techniques are

1. Gram stain

2. Acid fast stain

Gram stain 

The Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology. It is an example of differential staining—procedures that are used to distinguish organisms based on their staining properties.

Gram stain divides Bacteria into two classes— gram negative and gram positive.

Gram-staining procedure

 In the first step, the smear is stained with the basic dye crystal violet, the primary stain (1 minute). This is followed by treatment with Gram’s iodine solution (1 minute) which act as a mordant. The iodine increases the affinity of the cell to the dye so that the cell is stained more strongly.

The smear is next decolorized by washing with decolorizing agent, ethanol or acetone (30 seconds). This step is crucial and results in the differential aspect of the Gram stain.  Gram-positive bacteria retain the crystal violet, whereas gram-negative bacteria lose their crystal violet and become colorless. Finally, the smear is counterstained with a simple, basic dye different in color from crystal violet (1 minute). Safranin, the most common counterstain, colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple.

Mechanism of Gram Staining

The principle of Gram stain is based on the differences in bacterial cellwall

 

The difference between gram-positive and gram-negative bacteria is due to the physical nature of their cell walls.

Gram positive bacteria have a thick peptidoglycan layer whereas Gram negative bacteria has a thin peptidoglycan layer in their cellwall. There is an outer membrane, made of lipopolysaccharides in Gram negative cell wall.

When gram-positive bacteria then are decolorized with ethanol, the alcohol shrinks the pores of the thick peptidoglycan. Thus, the dye-iodine (CV-I) complex is retained during the short decolorization step and the bacteria remain purple. In contrast, gram-negative peptidoglycan is very thin and not as highly cross-linked. Alcohol treatment dissolves lipid from the gram- negative wall and increase its porosity. Duirng decolorization, purple crystal violet-iodine complex escapes through the pores formed, they then absorb the colour of counter stain – Safranin. 

Thus, Gram-positive bacteria stain purple, whereas gram-negative bacteria colour pink or red.

Critical points

The ability to resist decolorization is related to the chemical composition and structure of cell wall.

Grams iodine is a mordant that increase the affinity of the primary stain to the bacterial cells.

The decolorization step is the critical step that differentiate the bacteria. 

 

Acid-fast staining

It is a differential stain that is used in the identification of Mycobacterium - Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and leprosy, respectively. These bacteria have cell walls with high lipid content; in particular, mycolic acids—a group of branched-chain hydroxy lipids, which prevent dyes from readily binding to the cells.

Acid – fast staining procedure also called the Ziehl-Neelson technique, which uses heat and phenol to stain the cells with Carbol fuchsin. Once Carbol fuchsin has penetrated, M. tuberculosis and M. leprae are not easily decolorized by acidified alcohol (acid-alcohol), and thus are said to be acid-fast. Nonacid- fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.,

The acid-fast bacilli will stain bright red, and other cells will stain blue.

Procedure:

1. Prepare fixed smear of Mycobacterium on the slide.

2. Flood the smear with concentrated carbol fuchsin with flaming until steaming for (5 minutes), wash with water.

3. Add the decolorizer acid alcohol (20% H₂SO₄ in ethanol or 3% HCL in ethanol), until no more color appears, Wash with water.

4. Flood with methylene blue for (1 minute), Wash with water.

5. Dry the slide and observe under oil immersion.

Acid fast bacteria appear red and non-acid fast bacteria appear blue.

Acid- fastness is the ability of a microorganism to resist decolorization by acid alcohol after primary staining. It is due to the presence of a high lipid content (40-60%), in particular, mycolic acids, in the cell envelope.

(Gram negative bacteria have no more than (20%) while Gram positive bacteria have (1-4%) lipid in their cell envelope).

 

STAINING OF SPECIMENS

 Living microorganisms can be directly examined with the light microscope, but they need fixing and staining to increase visibility, highlight specific morphological features, and preserve them for future study.

The first step in most bacterial staining procedures is the preparation of a smear.

Smear Preparation

Smear preparation is a process in which bacterial culture is spread uniformly or as a thin layer on glass slide.

Bacterial Smear Preparation

The crucial step in staining is the preparation of a good smear. A good smear is important to get a good staining result.

Requirements:

• Culture of Bacteria

• Glass slides

• Bunsen burner

• Inoculating loop

• Glassware marking pencil/ Permanent Marker

Step I: Preparation of the glass slide:

• Clean, grease free slides are needed for smear preparation.

• Grease or oil from the fingers on slides must be removed by washing the slides with soap and water

• Finally rinse the slide with 95% alcohol and dry it.

 • Hold the slide by their edge.

Step II: Labeling of slides:

• Proper labelling of the slide is essential- every slide should be labelled clearly.

• A lead pencil /permanent CD marker is used to write on the one end of the glass slide.

 Step III: Preparation of smear:

• An evenly spread smear should be prepared covering area of 15-20mm diameter.

• Avoid thick and dense smear because thick smear prevent light penetration to visualize the morphology of cell.

• A good smear is one that, when dried, appears as a thin whitish layer or film. The print of textbook should be legible through the smear.

Different techniques are used for smear preparation depending upon culture media

i. Broth cultures (liquid medium)

• Re-Suspend the culture by tapping the tube with your finger.

• Depending on the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly over an area.

• Allow the smear to air-dry

ii. Culture plates (Solid medium)

• Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the loop. This helps to avoid cell clumping.

• The finished smear should occupy an area about the size of a nickel and should appear as a translucent, or semitransparent, whitish film

• Place a drop of water into the circle that has been created on the slide.

• Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony from the culture palte.

• Gently mix the bacteria into the water drop.

Step IV: Air dry

• Smear should be allowed to dry completely at room temperature at safe place

  Step V: Fixation of smear:

• The purpose of fixation of smear is to preserve and prevent smear being washed away during staining.

• Heat fixation - After smear is air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner or sprit lamp.  Avoid too much heating.

• After heat fix, allow the smear to cool before staining.

 

Fixation

The stained cells seen in a microscope should resemble living cells as closely as possible.

Fixation is the process by which the internal and external structures of cells and microorganisms are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microorganism usually is killed and attached firmly to the microscope slide during fixation.

There are two fundamentally different types of fixation.

Heat fixation is routinely used to observe procaryotes. Typically, a film of cells (a smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology but not structures within cells.

Chemical fixation is used to protect fine cellular substructure and the morphology of  delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, making them inactive, insoluble, and immobile. Common fixative mixtures contain components such as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.